When lights strike a surface, some lights bounce back and others are absorbed by or transmitted through the media underneath the surface. The light bounced off is reflection. The properties of light reflected, transmitted, and absorbed can be used to measure analyte concentrations.
Many medical instruments use the reflectance principle to detect analytes in a sample, for example the glucose meter from LifeScan, Abbott and Roche; blood analysis and monitoring products by Diavant Reflotron Plus; fructosamine measurement from LXN, while the lipid panel detection system from Cholestech. Reflectance biosensor takes readings from a surface that is covered by an inert porous matrix impregnated with a reagent that interacts with the analyte to produce a light-absorbing product. The operating principle of reflectance detection is as follows: A dry reagent is either immobilized or simply absorbed on an opaque membrane. The reagent reacts with an analyte, giving a product that absorbs the light of certain wavelength. When light strikes on the membrane, the unabsorbed light reflects back to the receiver, yielding an analysis of the properties sought. The amount of lights reflected is inversely proportional to the amount of analytes on the membrane.
Reflectance detection can be used not only in measuring analytes by chemical reaction, but also in immunoassays. In a solid phase competitive immunoassay, an antibody or antigen is immobilized on the membrane. Labeled analytes will compete with free analytes in the sample to bind to the membrane. In a solid phase sandwich assay, a labeled antibody is bound to an analyte, forming a complex, which in turn binds to a capture zone of the membrane where a specific antibody to the analyte is located. In both competitive and sandwich immunoassays, the analytes of interest and interferents are separated as the sample traverses the membrane. Unbound elements are washed through the membrane pores to the absorbent materials. The labeled analytes on the capture zone are readily detected in their natural state by the reflectance method.
Tests using the reflectance principle have several advantages. The most significant one is simplicity, multiple-step tests, like immunoassays, are simplified. The visible signal can be assessed without complicated operations. The tests for HCG, H. Pylori, drug screen, and HbA1c use this principle. These diagnostic tests are simple, quick and easy to use, and therefore have large market in home users and doctor's offices. The tests with reflectance principle, however do have disadvantaged due to the opaque membrane used. Light does not transmit through an ideal opaque membrane. In fact, it will reflect back almost completely to the receiver if no light absorbing product is generated from the analyte-substrate reaction on the membrane. A Thicker membrane absorbs more samples, thereby, increasing the analyte mass absorbed on the membrane, yielding more products, and absorbing more light, therefore less light reflecting back to the receiver. Based on this, increasing membrane thickness can enhance detection. Unfortunately, this potential advantage is negated by a physical limitation. Because of the opacity of the membrane, detection is possible only if light absorbent products are near the membrane surface. However the products are distributed evenly throughout the membrane depth. Any light absorbent products bound deeper than 10 μm under the surface become undetectable, their color being masked by the opaque membrane. Since the visible depth is constant for a given membrane, the impact of increasing the membrane thickness to improve sensitivity has limitations. It is for this reason that the majority of immunoassay tests using reflectance principle are not quantitative, but qualitative. A few immunoassays with reflectance principle are quantitative, but they have narrow separation range, limited sensitivity, and poor performance.
Analytes, such as glucose and cholesterol, have high concentrations in the blood. They range many fold from normal to abnormal levels. These analyte signals are sufficiently high to be measured by the reflectance detecting system, therefore, making sensitivity limitation not an issue in these tests. Unfortunately, in most clinical tests, including therapeutic drug monitoring, tumor markers, cardiac markers, hormones, infectious diseases indexes, autoimmune diseases indexes etc., analytes are low in concentrations. The concentration difference between normal and abnormal is marginal, especially in some drug monitoring tests, such as digitoxin and theophylline. The HbA1c test has a very narrow critical range to determine whether the patient's disease is under control. Some hormones (such as gonadotropines) fluctuate enormously and rapidly. Clearly, a quick, easy, accurate, and sensitive test with expanded testing range is needed for detecting such analytes.
To increase reflectance detecting sensitivity, many inventors use sensitive substrates to amplify the product color intensity through chemical reaction, while others improve the qualities of light sources and detecting systems. All these efforts do not significantly improve sensitivity. In addition, high quality light sources and detecting systems are costly. A system many folds more sensitive than that of current glucose or cholesterol detecting systems is needed for analytes with low concentration or those requiring an expanded separation range.
Tests requiring high sensitivity are usually run with a transmittance detecting system. Its principle is Beer's law. It states: A=αbc, where A is absorbance; α is the extinction coefficient absorbent product; b is length of the light path through the light absorbing product in the liquid solution (in cm); and c is the light absorbing product concentration (in moles per liter). Based on Beer's law, increasing the path length of the absorbing product will improve the detecting sensitivity. Transmittance detection is satisfactory for testing analytes with low concentration and narrow separation range, because it detects analytes in the full depth instead of a fraction layer near the surface as the reflectance detecting system does. When analyte concentrations are very low, the signals at different levels can not be distinguished by reflectance detection, but can be differentiated by transmittance detection if the light path through the analyte's product is increased.
Transmittance detecting principle has been widely used in many medical diagnostic systems, such as Roche's Fara/Fara II system, Abbott's Vision system, and Abbott Spectrum system. These systems have developed automated machines to run clinical tests. One example, ELISA (Enzyme-Linked Immuno-Sorbant Assay), is widely used in clinical immunoassays. These tests are performed in the liquid phase and in multiple steps. Technicians running a central clinical laboratory must be trained to operate these automated machines. Patients have to wait at least three to seven days to have testing result back. Promptly providing accurate information on diagnosis is crucial in treating acute diseases. Ideally, operation of the test should be simple, easy, no need trained laboratory technician to run the test. Reliable test results should be obtained in two or three minutes per parameter, so that the patient can be treated without delay. With such an efficient system, many tests currently run in central labs can be tested in small clinics or even at home. Patients will benefit tremendously from this convenience. Such applications will have a huge market. In this patent application, the invention uses a novel strategy to achieve this goal is described.